Table 1 Purification scheme developed for isolation of 3D6 and 4B3 dIgA.
From: 3D6 and 4B3: Recombinant expression of two anti-gp41 antibodies as dimeric and secretory IgA
STEP | EQUIPMENT | PROCEDURE | RECOVERY | |
|---|---|---|---|---|
| Â | Â | Â | 3D6 dIgA | 4B3 dIgA |
Ultra/Diafiltration | Kvick Start UDF Cassette, Millipore, 30 kD | Harvested cell culture supernatant containing dIgA is concentrated and buffer exchanged against PBS, pH 7.4 | >95 % | >95 % |
Lectin Affinity Chromatography | Immobilized Jacalin, Thermo Scientific | UDF retentate is immobilized onto a lectin affinity resin. After a washing step, bound product is eluted by applying 1.5 M D-galactose in PBS, pH 7.4 | 98.7 % | 106.1 % |
Ultra/Diafiltration | Kvick Start UDF Cassette, Millipore, 30 kD | The eluate is buffer exchanged against 20 mM Tris, 10 mM NaCl, pH 8.5 | >95 % | >95 % |
Anion Exchange Chromatography | DEAE Sepharose FF, GE Healthcare | The retentate is applied onto the anion exchanger. Post washing with 100mM NaCl product is eluted from the resin using 20 mM Tris, 200 mM NaCl, pH 8.5 | 88.4 % | 86.7 % |
Hydrophobic Interaction Chromatography | Phenyl-Sepharose 6FF low sub, GE Healthcare | (NH4)2SO4 is added to 0.75 M for 4B3-dIgA or 1.25 M for 3D6-dIgA to precipitate host cell proteins but not mAb itself. After a washing step 3D6-dIgA is eluted with 0.4 M (NH4)2SO4, which allows to isolate dimeric IgA from other IgA isoforms. 4B3-dIgA desorbs at 0 M (NH4)2SO4 | 44.1 % | 59.1 % |